We have previously provided conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle during their growth and development in the sand fly vector. In 2013, we have studied three main variables: the ability of diverse L. major strains from across its natural range to undergo mating with other L. major strains; the ability of L. major to mate across species with L. infantum; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. We have been able to demonstrate the sexual competency of L. major strains originating across the full geographic range of this parasite species, and in both natural and unnatural sand fly vectors. In crosses involving mating between L. major strains, hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable mating types that limit free genetic exchange. Remarkably, L. major was also able to mate across the species barrier with L. infantum, providing the first experimental support for the inter-species hybrid genotypes that have been described amongst natural isolates. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging in the posterior midgut well before the first appearance of infective stage, metacyclic promastigotes. Over 100 doubly-drug resistant hybrids were analyzed for DNA content and genotyped for parent alleles at a number of marker loci on different chromosomes, as well as the maxicircle DNA. For the L. major hybrids generated in the natural vector, 97% of the nuclear loci showed both parental alleles, suggesting that they are all full genomic hybrids; however, 3% of the marker loci showed only one parental allele, suggesting loss of heterozygosity by some unknown mechanism. Only uniparental inheritance of maxicircle kDNA was observed. These studies provide the strongest evidence to date that sex is a conserved and natural aspect of the reproductive strategy of Leishmania. We have identified the normal midgut microbiome as an essential factor controlling metacyclogenesis. L. major infected P. duboscqi sand flies treated with penicillin/streptomycin were completely inhibited in their ability to support the normal differentiation of promastigotes into the metacyclic stage. The bacterial diversity in normal and antibiotic treated flies was characterized by both culture dependent and independent methods by sequencing of the 16s rDNA. The possible roles of the normal bacterial communities in providing nutritional supplements essential to parasite differentiation, or to promoting nutrient deprivation or other stress conditions in the midgut required to trigger metacyclogenesis, will be investigated in future studies.